Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Patent
1996-04-08
1998-08-18
Naff, David M.
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
424 9351, 4352552, A01N 6300, C12N 114, C12N 116, C12N 118
Patent
active
057957711
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
Production of compounds of pharmaceutical significance by cultivation of recombinant yeasts is an expanding field of science and commerce. Purified recombinant hepatitis B surface antigen (HBSAg) is used as a vaccine for hepatitis B viral disease and is a well-known example of a pharmaceutically-significant recombinant protein.
Recombinant HBSAg is produced by fermentation of yeast in either complex or chemically-defined (synthetic) culture media. Generally, complex media, which contain crude sources of carbon and nitrogen such as yeast extract and peptones, support higher yields of cells and crude HBSAg than are achieved in synthetic media. However, fermentations performed in complex media are also more variable than fermentations which employ synthetic media. Inconsistencies in the fermentation adversely affect downstream purification steps and may also increase the cost of purified HBSAg.
Regulated expression systems are commonly used for the production of recombinant proteins. One type of regulated system provides tight nutritional control of the production of heterologous protein. This type of system maximizes biomass production and product stability while minimizing the adverse effects of heterologous protein expression on the host cell (Zabriskie et al., Enzyme Microbial Technol. 8:706-717 (1986)). Various components of the tightly-regulated galactose utilization pathway in yeast have been exploited successfully for controlled expression of a number of recombinant proteins, including portions of the hepatitis B envelope proteins (Carty et al., Biotech. Lett11:301-306, 1989)). Synthesis of proteins under the control of the GAL1 or GAL10 promoters occurs only in the presence of galactose and the absence of glucose (Oshima, 1981, Regulatory Circuits for Gene Expression: The Mechanism for Galactose and Phosphate, In: The Molecular Biology of the Yeast Saccharomyces, Vol. 1, pp. 159-180, Strathen, Jones, and Broach, (Eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Most regulated expression systems function in both complex and synthetic culture media.
It would be desirable to identify the component(s) of complex media that affect fermentation yields. It would also be desirable to determine a formulation of synthetic culture medium that supported the production of recombinant protein more nearly equivalent to that achieved in complex media. Advantages of such discoveries would include the establishment of a more reproducible fermentation process and a more predictable purification process.
YEHD medium is one of many complex media that is used for the production of recombinant HBSAg. YEHD contains (per liter of distilled water) 20 g yeast extract powder, 10 g soy peptone and 16 g glucose. The amount of crude HBSAg produced when recombinant yeast are cultivated in YEHD varies from fermentation to fermentation.
Preliminary studies identified yeast extract powder as the component of YEHD responsible for a major part of the variability in fermentation yields. Chemical analyses of different manufacturing lots of yeast extract powder showed that the concentrations of at least six components of yeast extract powder varied significantly between lots. Further analyses of additional lots of yeast extract powder identified choline as a component of yeast extract powder that was strongly correlated with the productivity of the fermentation.
These data were used in the development of a synthetic culture medium that supports the growth of yeast cells and the production of recombinant proteins. The synthetic culture medium of the present invention differs from other synthetic media used to cultivate recombinant yeast in that it contains choline. The culture medium of the present invention may be used to prepare stock cultures of recombinant strains of Saccharomvces cerevisiae, including but not limited to strains of S. cerevisiae that produce hepatitis B surface antigen. The culture medium of the present invention may also be used to produce crude recombinant proteins, includi
REFERENCES:
patent: 4855238 (1989-08-01), Gray et al.
Carty, et al., "Fermentation of Recombinant Yeast Producing Hepatatis B Surface Antigen", J. Ind. Microbio., vol. 2, pp. 117-121 (1987).
Zabriski, et al., "Factors Influencing Productivity of Fermentations Employing Recombinant Microorganisms", Enzyme Microb. Technol., vol. 8, pp. 706-171 (1986).
Jung, et al., "Supplement of Nutrients for Effective Cultivation of Hepatitis B Surface Antigen-Producing Recombinant Yeast", Biotechnol. Letters, vol. 13, pp. 857-862 (1991).
Oshima, et al., "Regulatory Circuits for Gene Expression: The Metabolism of Galactose and Phosphate in the Molecular Biology", vol. 1, pp. 159-180 (1981).
O'Connor, et al., "Design and Evaluation of Control Strategies for High Cell Density Fermentations", Biotechnol. and Bioengin., vol. 39, pp. 293-304 (1992).
Kitano, et al., "Recombinant Hepatitis B Virus Surface Antigen P31 Accumulates as Particles in Saccharomyces cerevisiae", Biotechnol., vol. 5, pp. 281-283 (1987).
Li, et a., "Hyper-resistance to Nitrogen Mustard in Saccharomyces cerevisiae is Caused by Defective Choline Transport", Curr. Genet., vol. 19, pp. 423-427 (1991).
Bailis, et al., "Cis and Trans Regulatory Elements Required for Regulation of the CHO1 Gene of Saccharomyces cerevisiae", Nucleic Acids Red., vol. 20, No. 6, pp. 1411-1418 (1992).
ATCC Catalogue of Funji Yeasts, 17th Edition, pp. 412-413 (1987).
George Hugh
Herber Wayne K.
Merck & Co. , Inc.
Naff David M.
Tribble Jack L.
Ware Deborah K.
Yablonsky Michael D.
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